Human Defensin DEF-X

ABSTRACT

The present invention relates to a human polypeptide defensin Def-X, homologous to HNP-4, its genomic DNA and cDNA, vectors, cells transformed with said vectors, the use of said polypeptides as antibiotic, cytotoxic, repair and endocrine regulatory agent and as pesticide as well as cosmetic or pharmaceutical compositions for the treatment of microbial infections, in particular bacterial, fungal and viral infections, or parasitic infections, cancers, of inflammation and of immune deficiency. Finally, the invention comprises diagnostic methods and kits for the determination of a microbial or parasitic infection and of an inflammation, or for screening for predisposition to immune deficiencies or cancer diseases.

This application claims priority, under 35 U.S.C. § 371, toPCT/FR98/01864.

The present invention relates to a novel human polypeptide defensinDef-X, homologous to HNP-4, its genomic DNA and CDNA.

The invention also relates to cloning and expression vectors, and cellstransformed with said vectors. The subject of the invention is also theuse of said polypeptides as antibiotic, cytotoxic, repair and endocrineregulatory agent and as pesticide as well as cosmetic or pharmaceuticalcompositions for the treatment of microbial infections, in particularbacterial, fungal and viral infections, or parasitic infections,cancers, of inflammation and of immune deficiency. Finally, theinvention comprises diagnostic methods and kits for the determination ofa microbial or parasitic infection and of an inflammation, or forscreening for predisposition to immune deficiencies or cancer diseases.

Antimicrobial substances are key elements in the defence ofmulticellular organisms. Among these substances, there are both simpleinorganic compounds (hydrogen peroxide, hypochlorous acid, nitric oxide)and complex proteins and peptides. They are present at the first linesof defence, at the surface of the mucous membranes of various organs, inparticular in the epithelial cells of the intestine and of the lungs,depending on the species, as well as in the microbicidal organs ofphagocytic cells of hematopoietic origin, where they were firstidentified. Their synthesis de novo or their release from storagesites—organelles of the lysosome or cytoplasmic granule type which arecapable of storing them in an inactive or latent form—can be inducedrapidly, which makes them particularly important in the early phases ofresistance to infections (Martin et al., 1995).

The antimicrobial proteins of less than one hundred aminoacids in sizeare arbitrarily called antimicrobial peptides. Several families ofantimicrobial peptides have been identified, which differ in thepresence within them of disulfide bridges, in their aminoacidcomposition, their structural conformation and in their activityspectrum. The antimicrobial peptides comprising six conserved cysteinesform the defensin family. This family is composed of antimicrobialpeptides which are present in numerous species, which are abundant andwhich are about 3-4 kDa (Ganz and Lehrer, 1994). These peptides areformed of 30 to 40 aminoacids, of which six invariant cysteins whichform three intramolecular disulfide linkages. They have complexconformation, are amphipathic, rich in beta antiparallel sheets but lackalpha helices (Lehrer and Ganz, 1992). The antimicrobial action ofdefensins is thought to result from their insertion into the membranesof the target cells, allowing the formation of voltage-dependentchannels. White et al. (1995) describe the possible mechanisms ofmembrane insertion and of formation of multimeric pores by thedefensins, which allow the permeabilization of the membranes of thetarget cells, for example microbial or tumor cells. The crystallographicstructure of human neutrophil defensin HNP-3 (see below) has beendetermined, and a specific mechanism of dimerization of the humanneutrophil defensins is in addition suggested. Increased knowledge ofthis family of peptides and comparison of their sequences and activityspectra will make it possible to better understand these mechanisms andtheir specificities, as well as the aminoacid residues more particularlyinvolved in these phenomena.

The defensins are divided into three familities of peptides which arestructurally different: the “conventional” defensins, the beta-defensinsand the insect defensins. These families exhibit differences as regardsthe position of and the distance between the conserved cysteineresidues, as well as those of other conserved aminoacids (proline,glycine) (Ganz and Lehrer, 1995).

Human defensins, of the conventional type, come essentially from twosources. They were first identified by peptide purification fromneutrophil extracts. Four defensins have thus been isolated: “humanneutrophil peptides” HNP-1, HNP-2, HNP-3, and HNP-4. The first three aredifferent products of the same gene (Ganz et Lehrer, 1995). These threepeptides represent 99% of the defensin content of the neutrophils,whereas HNP-4 is also present therein, but at concentrations which are100 times lower. More recently, two human enteric defensins, HD-5 andHD-6, were characterized in the small intestine and more precisely inthe Paneth cells (Bevins et al., 1996). While 16 enteric defensin geneshave been identified in mice, only these two homologs have beenidentified in humans (Mallow et al., 1996).

Defensins have an antimicrobial action on a broad spectrum ofmicroorganismes in vitro (Martin et al., 1995). This activity spectrum,which is particularly broad, comprises bacteria, Gram-positive andGram-negative bacteria, several fungi, mycobacteria, parasites includingspirochetes and several enveloped viruses including the HSV and HIVviruses. They are also cytotoxic for several categories of normal andmalignant cells, including cells resistant to TNF-alpha and to thecytolytic NK factor (Kagan et al., 1994). The large quantity of targetsof the defensins and their abundance in blood cells specialized in theimmune defence, as well as the dramatic increase in their concentrationduring severe infections, suggest that these molecules could play animportant role in the natural immunity to infections and to cancers. Inparticular, the increase in the transcription of the defensin genes andthe release of cytoplasmic granules containing presynthesized defensinsin response to stimuli, contributes to the local antimicrobial response,it being possible for the defensins to participate in the inflammatoryreaction, in the repair processes and in endocrine regulation duringinfection. The hematopoietic defensins could contribute to thephenomenon of lysis of cancer cells, a phenomenon which is mediated bythe neutrophils during the antibody-dependent immune response. Theprecise physiological role of the enteric defensins is not clearlyestablished. They could stem the proliferation of the intraluminal floraor prevent the translocation of bacteria across the intestinal mucosa(Mallow et al., 1996). The abundance of the defensin mRNA in the Panethcells reinforces the hypothesis that these epithelial cells could play akey role in the immune defence in the intestine. It has moreover beenshown that their expression scheme coincides with the appearance of thePaneth cells during embryogenesis. Mallow et al. (1996) have suggestedthat low levels of expression of enteric defensins in the fetus could bethe evidence of an immaturity of a local defence, which would predisposechildren born prematurely to infections due to intestinalmicroorganisms.

A defensin concentration corresponding to 10% of the normal level isobserved in patients suffering from “specific granule deficiency”, arare disease of the development of the granulocytes. The affectedsubjects suffer from frequent infections caused by common bacteria (Ganzand Lehrer, 1995).

Biochemically modified defensins are potential prophylactic andtherapeutic agents against infections (Ganz and Lehrer, 1995). Researchrelating to these antimicrobial peptides or other moleculesparticipating in the natural immunity have gained special importancesince phenomena of resistance of microorgnisms to traditionalantibiotics started to develop (Bevins et al., 1996).

The primary structure of defensins, in particular of human defensins,has been the subject of recent studies (White et al., 1995; Mallow etal., 1996). The conventional defensins comprise 29 to 35 aminoacids, butare derived from precursors—preproteins—comprising 90 to 100 aminoacids.The proteolytic maturation of the human neutrophil defensins to maturepeptides is coupled with their despatch to the granulocytes; thefunction of the propeptide would include the inactivation of theprecursor form of defensin and a support for the acquisition of theactive conformation of the mature peptide (Martin et al., 1995). Thepeptide homologies are maximal at the level of the signal peptides, andminimal at the level of the mature peptides, which comprise neverthelesssix cysteine residues which are fully conserved. While the conservationof these residues appears to be necessary for the acquisition ofsecondary structures which are involved in the activity of defensins,the differences in sequences which exist within the very large family ofthese antimicrobial peptides, in particular at their N-terminal end, butalso in other nonconserved regions, appear to be important determinantsof their activity spectrum, and of their antimicrobial or cytotoxicefficacy. The identification of novel members of this family ofpeptides, in particular of human defensins, is therefore necessary forunderstanding their mechanism of action and their specificity as well asfor their use as anti-infectious and/or cytotoxic agents, or fordesigning variant peptides exhibiting specific spectra and/or of reducedor increased efficacy.

Sparkes et al. (1989) located the gene encoding HNP-1 on chromosome 8,in the 8p23 region. Bevins et al. (1995), and Mallow et al. (1996)Icoated the two genes encoding HD-5 and HD-6 on chromosome 8, moreprecisely in the 8p21-pter region, a region including the regionpreviously identified as carrying the hematopoietic defensins. The genesencoding the human enteric defensins HD-5 and HD-6 contain two exons,whereas those encoding the hematopoietic defensins contain three ofthem, the last two exons encoding the prepropeptide, both in humans andin guinea pigs and rabbits (Mallow et al., 1996). Comparison of thegenome sequences of the HD-5 and HD-6 genes has revealed a very strongsimilarity between the noncoding flanking sequences in 5′, suggestingthat the latter contain the information necessary to the tissuespecificity of the expression of these genes; these same regions carry,in addition, many binding sites for transcription factors, including twoAP2 sites and six IL6 sites, suggesting pathways for regulating theexpression of these genes during inflammatory processes. More generally,the very high degree of similarity between the sequences and the genomicorganization of the defensins HNP-1, 2, 3, 4 and HD-5 and 6 led Bevinset al. (1995) to an evolutionary model attempting to relate thechromosomal organization of the family, and the homologous fractions ofeach pair of genes.

It is finally advantageous to note that the chromosomal region 8p23 isinvolved in numerous pathologies, in particular cancer pathologies:there may be mentioned, for example, hepatocellular carcinoma (Becker etal., 1996), non-small cell lung cancer (Sundareshan and Augustus, 1996),prostate cancer (Ichikawa et al., 1996), and colorectal carcinome(Yaremko et al., 1994). Although this has never been documented, it ispossible that a deficiency in either of the human defensins has a rolein the predisposition to such pathologies, or in their development.

The present invention relates to a novel human defensin, Def-X, which ishomologous to defensin HNP-4.

The subject of the present invention is therefore an isolatedpolypeptide chosen from the following polypeptides:

a) polypeptide whose amino acid sequence is the sequence SEQ ID No. 3;

b) homologous, variant or modified polypeptide of the polypeptide whoseamino acid sequence is the sequence SEQ ID No. 3;

c) polypeptide whose amino acid sequence is the amino acid sequence of abiologically active fragment of a polypeptide as defined in a) or b);

d) polypeptide comprising at least one fragment as defined in c).

In the present description, <<polypeptide>> will also be intended todesignate a proteine or a peptide.

According to a preferred embodiment, the polypeptide according to theinvention is characterized in that it consists of at least one of thefollowing fragments:

a) signal peptide whose aminoacid sequence is the sequence SEQ ID No. 4,corresponding to the sequence between position 1 and position 19, endsincluded, of the aminoacid sequence SEQ ID No. 3;

b) proregion whose aminoacid sequence is the sequence SEQ ID No. 5,corresponding to the sequence between position 20 and position 63, endsincluded, of the aminoacid sequence SEQ ID No. 3;

c) mature peptide whose aminoacid sequence is the sequence SEQ ID No. 6,corresponding to the sequence between position 64 and position 94, endsincluded, of the aminoacid sequence SEQ ID No. 3; or

d) homologous, variant or modified fragment of a peptide according toa), b) or c).

Still preferably, the polypeptides according to the present inventioncorrespond to the primary structure of the mature defensin definedabove, that is to say the structure corresponding to the followingaminoacid sequence SEQ ID No. 6:

Ile Cys His Cys Arg Val Leu Tyr Cys Ile Phe Gly Glu His Leu Gly Gly ThrCys Phe Ile Leu Gly Glu Arg Tyr Pro Ile Cys Cys Tyr

its homologs, variants or modified forms as well as their biologicallyactive fragments and the polypeptides containing them.

It is clearly understood that the polypeptides of the invention are in anonnatural form, that is to say that they are not taken in their naturalenvironment but that they may have been obtained by purification fromnatural sources or obtained by genetic recombination or by chemicalsynthesis as will be described below.

<<Homologous polypeptide>> is understood to mean a polypeptide whoseaminoacid sequence exhibits at least 80%, and preferably 90%, ofaminoacids in common.

<<Variant polypeptide>> is intended to designate a mutated polypeptideor a polypeptide corresponding to a polymorphism which may exist, inparticular in human beings and which may exhibit a truncation, asubstitution, a deletion and/or an addition of at least one aminoacidcompared with the polypeptide according to the invention.

<<Modified polypeptide>> is understood to designate a polypeptideobtained by genetic recombination or by chemical synthesis as will bedescribed below, exhibiting a modification relative to the normalsequence. These modifications may in particular apply to the pre, pro-or mature domains of the polypeptide according to the invention, theaminoacids responsible for a specificity of spectrum or of efficacy ofactivity, or responsible for the structural conformation, the charge orthe hydrophobicity, and the multimerization and membrane insertion ofthe polypeptide according to the invention. It is thus possible tocreate polypeptides with equivalent, increased or reduced activity, andwith equivalent, lower or broader specificity. The modifications mayalso apply to the sequences involved in the maturation, transport andaddressing of the polypeptide.

<<Biologically active fragment>> of a polypeptide according to theinvention is intended to designate a polypeptide fragment which hasconserved at least one activity of the polypeptide from which it isderived, in particular:

capable of being recognized by an antibody specific for a polypeptideaccording to the invention; and/or

capable of acting as an antibiotic; and/or

capable of acting as an cytotoxic agent; and/or

capable of acting as an antitumor agent; and/or

capable of modulating tissue repair, endocrine regulation or theinflammatory process, in particular during an infection.

According to the invention, the biologically active fragments of thepolypeptides according to the invention will have a minimum of 10 aminoacids, preferably 15 amino acids.

As has been indicated above, among the biologically active fragments, apreferred fragment is the mature peptide having the aminoacid sequenceSEQ ID No. 6.

Among the homologs of the mature peptide, there should be mentioned thepolypeptides in which up to 5 amino acids have been modified, truncatedat the N- or C-terminal end, or deleted, or added, which representsabout 80% of the sequence.

The biologically active fragments of this mature peptide preferablycomprise from 10 to 15 aminoacids, the advantage of which may be beingable to be easily obtained by chemical synthesis.

As indicated, the objective of the modifications of the maturepolypeptide will be in particular:

to modulate the activity of the defensin,

to modify its specificity, both at the level of the microorganisms onwhich it is active and on its tissue localization,

to modify its bioavailability.

The preceding compounds may be obtained using combinatorial chemistry,in which it is possible to systematically vary portions of thepolypeptide before testing them on models, cell cultures ormicroorganisms, for example, in order to select the compounds which aremost active or which have the desired properties.

Chemical synthesis also has the advantage of being able to use:

nonnatural aminoacids, or

nonpeptide bonds.

Thus, to enhance the lifespan of the peptides, it may be advantageous touse nonnatural aminoacids, for example in D form, or aminoacid analogs,in particular sulfur-containing forms for example.

Finally, the structure of the mature defensin or of its homologs,variants or modified forms, as well as the corresponding fragments, maybe integrated into chemical structures of the polypeptide type and thelike. Thus, it may be advantageous to provide at the N- and C-terminalends compounds not recognized by proteases.

The invention also comprises the nucleic acids encoding a polypeptideaccording to the invention.

According to a preferred embodiment, the nucleic acids according to theinvention will be chosen from the following nucleic acids:

a) nucleic acid having the sequence SEQ ID No. 1 (genomic);

b) nucleic acid having the sequence SEQ ID No. 2 (cDNA);

c) equivalent, homologous, mutated or modified nucleic acid, comparedwith the nucleic acids according to a) or b);

d) fragments of the sequences a), b) or c) having at least ten bases;

e) nucleic acid capable of hybridizing with one of the sequences asdefined in a), b), c) or d).

It is understood that the present invention does not relate to thegenomic sequences in their natural chromosomal environment; they aresequences which have been isolated, that is to say that they have beenrecovered directly or indirectly, their environment having been at leastpartially modified.

They may thus be genomic DNA, cDNA or RNA comprising nonnaturalnucleotides or not; they may also be isolated natural nucleic acids, orsynthetic nucleic acids.

Equivalent nucleic acid will be understood to mean a nucleic acidencoding the polypeptides according to the invention, taking intoaccount the degeneracy of the genetic code, and the corresponding cDNAsand RNAs.

Homologous nucleic acids will be understood to mean nucleic acid whosesequence exhibits at least 80%, preferably 90%, homology with thenucleic sequences according to the invention.

Mutated nucleic acid will be understood to mean any nucleic acidencoding a variant polypeptide according to the invention, and anynucleic acid comprising, compared with the sequences SEQ ID No. 1 andSEQ ID No. 2, at least one mutation in the promoter and/or regulatorysequences which may have an effect on the expression of the polypeptide,in particular on its level of expression and the tissue-specificitythereof. The sequences exhibiting a polymorphism which is present inhuman beings are therefore included in the invention. Among thesepolymorphisms, some may lead to immune deficiencies, in the response toinfections, to predispositions to and/or to the development of cancers.

Modified nucleic acid will be understood to mean any nucleic acidencoding a modified polypeptide according to the invention, or anynucleic acid obtained by mutagenesis according to techniques well knownto persons skilled in the art, and comprising modifications relative tothe normal sequences, in particular mutations in the regulatory and/orpromoter sequences, in particular leading to a modification in the leveland/or the tissue-specificity of the expression of the polypeptide.

The present invention relates to all the primers and probes, which maybe labeled according to methods well known to persons skilled in theart, which make it possible to identify, in particular by techniquesbased on hybridization or on amplification, for example by PCR, thenucleic sequences according to the invention, including discriminatingbetween the normal sequences and the mutated sequences.

Among the nucleic acid fragments of interest, there should be mentionedin particular the antisense oligonucleotides, that is to say whosestructure ensures, by hybridization with the target sequence, inhibitionof the expression of the corresponding product. The senseoligonucleotides should also be mentioned which, by interacting withproteins involved in the regulation of the expression of thecorresponding product, will induce either an inhibition, or anactivation of this expression.

They may be sequences which act both at the level of the exon or intronsequences described and on the flanking sequences, in particular thepromoters and/or 5′ UTR regions.

The present invention also relates to cloning or expression vectorscomprising a nucleotide sequence as described above.

These cloning or expression vectors may comprise elements ensuringexpression of the sequence in a host cell, in particular promotersequences and regulatory sequences which are effective in said cell.

It being possible for the vector in question to be autonomouslyreplicating or to be intended to ensure the integration of the sequencewithin the chromosomes of the host cell.

In the case of autonomously-replicating systems, depending on the hostcell, either prokaryotic or eukaryotic, systems of the plasmid type orviral systems will be preferably used, it being possible for the vectorviruses to be in particular advenoviruses (Perricaudet et al., 1992),retroviruses, poxviruses or herpesviruses (Epstein et al., 1992).Persons skilled in the art know the technologies which can be used foreach of these viruses.

Thus, it is known to use, as viral vector, defective viruses whoseculture is carried out in complementation cells, this avoiding thepossible risks of prolifeation of an infectious viral vector.

When the integration of the sequence into the chromosomes of the hostcell will be desired, it will be necessary to provide, on either side ofthe nucleotide sequence to be integrated, one or more sequences derivedfrom the host cell in order to ensure the recombination. These are alsomethods which are widely described in the prior art. It will bepossible, for example, to use systems of the plasmid or viral type;

such viruses will be, for example, retroviruses (Temin, 1986) or theAAVs, Adenovirus-Associated Virus (Carter, 1993).

The invention also relates to the prokaryotic or eukaryotic cellstransformed with a vector as described above in order to ensure theexpression of a natural, normal or variant, or modified defensin Def-X,or for example, of one of its fragments.

As indicated above, the present invention also relates to thepolypeptides obtained by culturing the cells thus transformed andrecovering the protein expressed, it being possible for said recovery tobe carried out intracellularly or extracellularly in the culture mediumwhen the vector has been designed to ensure the secretion of the proteinby means, for example, of a “signal” sequence, the polypeptide being inthe form of a pre-polypeptide or prepro-polypeptide. The constructsallowing the secretion of the polypeptides are known, both forprokaryotic systems and for eukaryotic systems. In the context of thepresent invention, some of the polypeptides Def-X may comprise their ownsecretory or membrane insertion system.

It is clearly understood that the recombinant polypeptides according tothe invention may be obtained in glycosylated or nonglycosylated formand may have the natural tertiary structure or not.

Among the cells which can be used for the production of thesepolypeptides, there should of course be mentioned bacterial cells (Olinsand Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animalcells, in particular mammalian cell cultures (Edwards and Aruffo, 1993)but also insect cells in which methods using baculoviruses, for example(Luckow, 1993), may be used.

The cells thus obtained can make it possible to prepare natural, variantor modified polypeptides, Def-X, but also fragments of thesepolypeptides, in particular polypeptides which may correspond to thebiologically active fragments.

The present invention relates, in addition, to the same polypeptidesaccording to the invention but which are obtained by chemical synthesisand which may comprise nonnatural or modified aminoacids.

The polypeptides according to the present invention, in particularmature defensin, as well as the homologs, derivatives or modified maturepolypeptides, may be obtained by chemical synthesis using any one of thenumerous peptide syntheses known, for example the techniques using solidphases or techniques using partial solid phases, by condensation offragments or by a conventional synthesis in solution.

When the compounds according to the present invention are synthesized bythe solid phase method, the C-terminal aminoacid is attached to an inertsolid support and comprises groups protecting its amino group at thealpha position (and if necessary, protections on its functional sidegroups).

At the end of this step, the group for protecting the amino terminalgroup is removed and the second aminoacid, which also comprises thenecessary protection, is attached.

The N-terminal protecting groups are removed after each aminoacid hasbeen attached; on the other hand, the protection is of course maintainedon the side chains.

When the polypeptide chain is complete, the peptide is cleaved from itssupport and the protective side groups are removed.

The solid phase synthesis technique is described in particular inStewart et al. (1984) and Bodanszky (1984).

The details of the synthesis will not be mentioned here; it shouldsimply be recalled that the protective groups which are preferred forthe alpha-amino groups are protective groups of the urethane type (BOCor FMOC). As regards the coupling reagents, they are very numerous;among them, there should of course be mentioned more particularlyN,N′-diisopropylcarbodiimine (DIC) which is used in general in DMF orDCM.

When it will be desirable to use nonnatural aminoacids, it may benecessary to provide other types of reagent and in particular othertypes of protection system.

The present invention also relates to polyclonal or monoclonalantibodies obtained by immunological reaction in a human or animal bodywith an immunogenic agent consisting of a polypeptide according to theinvention, in particular a polypeptide obtained by culturing one of thecells which have just been described, or by chemical synthesis asindicated above.

The invention therefore extends to the monoclonal and polyclonalantibodies or one of their fragments, chimeric antibodies, which arecapable of specifically recognizing a polypeptide according to theinvention.

The invention also comprises the antibodies according to the invention,characterized in that they labeled.

The labeled antibodies may be, for example, immunoconjugated withenzymes such as peroxidase or alkaline phosphatase, or labeled with theaid of fluorescent compounds, biotin or radiolabeled. The labelingtechniques are well known to persons skilled in the art and will not bedeveloped in the present description.

The invention also extends to the use of a polypeptide according to theinvention as antimicrobial, in particular antibacterial, antifungal,antiviral and/or antiparasitic agent, as cytotoxic agent, in particularfor anticancer use, and/or as agent for modulating inflammatory, tissuerepair and endocrine, in particular corticostatic, regulating processes.

According to another aspect, the invention relates to a pharmaceuticalcomposition comprising a polypeptide according to the invention, whichmay be combined with a pharmaceutically acceptable vehicle.

Such a composition may be administered by the systemic, local or topicalroute.

Its mode of administration, its dosage, its optimal galenic forms may bedetermined according to the criteria generally taken into account inestablishing a treatment appropriate for a patient, in particular theirage, their body weight, tolerance of treatment, its observed sideeffects, and the like.

The invention also comprises a pharmaceutical composition comprising avector according to the invention which is capable of expressing in vivoa polypeptide according to the invention, which may be combined with apharmaceutically acceptable vehicle.

It is also possible to envisage the expression of polypeptides or theirfragments in vivo, in particular by means of gene therapy and using thevectors which were described above.

In the context of gene therapy, it is also possible to envisage the useof the sequences of the genes or of the cDNAs described above,<<naked>>, this technique was in particular developed by the companyVical, which has shown that it was, under these conditions, possible toexpress the polypeptide in some tissues without having recourse to thesupport of a viral vector in particular.

Still in the context of gene therapy, it is also possible to envisagethe use of cells transformed ex vivo, which cells may then bereimplanted, either as such, or inside systems of the organoid type, asis also known in the state of the art (Danos et al., 1993). It is alsopossible to envisage the use of agents facilitating the targeting of adefined cell type, penetration into the cells or transport to thenucleus.

Said pharmaceutical compositions are, according to the invention,intended for the prevention and/or treatment of microbial infections, inparticular microbial infections of bacterial, Gram-positive orGram-negative bacteria, mycobacterial, fungal and viral origin, orparasitic, in particular spirochet, infections.

According to a preferred embodiment, the invention advantageouslyrelates to the pharmaceutical compositions according to the invention,characterized in that the viral infections are infections linked toenveloped viruses, in particular the HSV and HIV viruses.

The subject of the invention is also pharmaceutical compositionsaccording to the invention, intended for the prevention and/or treatmentof cancers, in particular melanomas, liver, prostate or non-small celllung cancer or colorectal carcinoma.

The invention comprises, in addition, pharmaceutical compositionsaccording to the invention, intended to increase the immune defenses, toincrease the immune defenses in the case of acquired immunodeficiency orto prevent the immunodeficiency, in particular for the treatment ofpsoriasis, or to modulate the inflammatory processes in cases inparticular of chronic inflammatory diseases.

The polypeptides according to the present invention can be moreparticularly used in external topical form, for example on the skin andthe mucous membranes. These external topical forms may be forpharmaceutical, dermatological or cosmetic use.

In particular, these compositions may be used as pharmaceuticalantiseptic agent or as antiseptic in some cosmetics, either forcleansing the skin or superficial body growths and/or as preservativefor the compositions.

The topical compositions according to the present invention may be usedin particular in some skin, eye, vaginal or buccal conditions. They mayalso be used as additional cosmetic agent, in particular in sometreatment shampoos.

The invention also relates to the detection of the absence or of anabnormal quantity of protein or of nucleic acid corresponding todefensin X as marker of an infection or of pathologies which will bedescribed below.

The invention also relates to the detection of an abnormal form of theprotein or the presence of an abnormal nucleic acid corresponding to amutated defensin which may possibly be completely inactive. In thiscase, the presence of this abnormal form may be a marker ofpredisposition to certain conditions, in particular immunodeficiencyand/or cancers.

Accordingly, the present invention relates to a method of diagnosing ofan immunodeficiency and/or of a predisposition to certain types ofcancer, characterized in that the presence of an abnormal defensinand/or of a sequence encoding an abnormal defensin is detected in asample from a patient.

The diagnostic methods according to the present invention allow inparticular the detection of an immunodeficiency and/or of apredisposition to one or more cancers, in particular those cited above,in particular in at-risk families. This type of diagnosis will ingeneral be carried out by the detection of the mutated forms of theprotein or of the nucleic acid sequences.

However, the invention also relates to methods for the diagnosis ofinflammation, immunodeficiency, predisposition to conditions of thecancer type and/or infections due to microorganisms or linked to animmune deficiency or inflammatory phenomenon, characterized in that theycomprise assaying a polypeptide or a nucleic acid according to theinvention in a biological sample and comparing the result of said assaywhich is obtained with the quantity of polypeptide or nucleic acidnormally present in an equivalent biological sample.

In this case, the peptide assay will allow, in general, detection of amicrobial or parasitic infection and/or of an inflammation. The peptideassays may be carried out by any known method, ELISA or RIA for example.The detection of an abnormal form of defensin-X may be carried out, forexample, with the aid of a monoclonal antibody which is specific forthis form, in particular the antibodies which are the subject of theinvention.

According to a preferred embodiment, the invention advantageouslycomprises the methods characterized in that they use an oligonucleotideprobe and/or primer according to the invention.

The methods in which all or part of the sequence corresponding to thepolypeptide Def-X is amplified beforehand by assaying nucleic acidaccording to the invention will be generally preferred, it beingpossible for these amplification methods to be carried out by theso-called PCR or PCR-like methods. PCR-like will be understood todesignate all the methods using direct or indirect reproductions of thenucleic acid sequences, or in which the labeling systems have beenamplified, these techniques are of course known; in general, theyinvolve the amplification of DNA by a polymerase; when the originalsample is an RNA, it is advisable to carry out a reverse transcriptionbeforehand. There are currently very numerous methods which allow thisamplification, for example the so-called NASBA “Nucleic Acid SequenceBased Amplification” (Compton, 1991), TAS “Transcription basedAmplification System” (Guatelli et al., 1990), LCR “Ligase ChainReaction” (Landegren et al., 1988), “Endo Run Amplification” (ERA),“Cycling Probe Reaction” (CPR), and SDA “Strand DisplacementAmplification” (Walker et al., 1992), methods which are well known topersons skilled in the art.

The invention relates, in addition, to diagnostic kits or boxes for thedetermination of a microbial or parasitic infection, an inflammation, animmunodeficiency and/or a predisposition to cancer-type conditions,characterized in that they comprise an antibody according to theinvention.

The diagnostic kits or boxes for the determination of a microbial orparasitic infection, an inflammation, an immunodeficiency and/orpredisposition to cancer-type conditions, characterized in that theycomprise a probe and/or a primer according to the invention are alsoincluded in the invention.

Finally, the subject of the invention is the use of a polypeptideaccording to the invention as pesticide, in particular for thecultivation of plants of industrial interest such as, for example, foodplants such as corn, wheat, soybean, rice or rape, fodder plants, fruittrees, grape vine or ornamental plants.

Other characteristics and advantages of the present invention willemerge on reading the examples below, illustrated by the figures whoselegends are described below.

Legend to the Figures

FIG. 1

Genomic sequence of hDef-X.

Presented is the entire genomic DNA sequence of hDef-X which exhibitssignificant homology with the gene encoding hDef-4 (HNP-4).

The sequence has the following sites, the presence of which is deducedby homology with the hDef-4 sequence:

CAAT box 1711-1714 TATA box 1758-1767 mRNA start 1836 exon 1 1836-1874splicing site 1 GTCAGT Alu insertion 2155-2335 L1 fragment insertion2710-2780 splicing site 2 CAG exon 2 3394-3577 start of coding phase3406 splicing site 3 GTGAGA splicing site 4 CAG exon 3 4164-4379 end ofcoding phase 4276 polyadenylation site 4374-4379.

FIG. 2

Alignment of the genomic sequences of the human defensins Def-X andDef-4 (HNP-4).

Alignment of the entire genomic DNA sequence of the novel defensin Def-Xexhibiting homology with the genomic DNA of hDef-4 (GenBank accessionnumber U18745).

The annotations present the positions on the hDef-4 sequence of thesignals CAAT box, TATA box, splicing sites, beginning and ends ofintrons/exons, start of transcription and polyadenylation site.

FIG. 3

Alignment of the cDNA sequences of hDef-4 (HNP-4) and hDef-X.

The sequences exhibit an overall homology of 61.4%. The alignmentreveals an insert of about 75 bases downstream of a STOP codon, whichare present on the sequence of hDef-4, but not on that of hDef-X; thestrong homology continues on the whole region between the end of thisinsert and that of the cDNA. Outside this insertion region, the degreeof homology between nucleic sequences is therefore remarkable.

FIG. 4

Peptide sequence of the protein hDef-X.

The position of the sites of cleavage of the signal peptide and of thepro region were deduced from the alignment of the peptide sequences ofhDef-4 and hDef-X.

FIG. 5

Alignment of the peptide sequences of the known human defensins hDef-1,hDef-4, hDef-5, and hDef-6 with hDef-X.

* The star indicates an amino acid which is conserved on the fivesequences.

• The dot indicates an amino acid whose class is conserved on the fivesequences (amino acid which is either identical, or which is the subjectof a conservative substitution).

{circumflex over ( )} six arrows indicate the positions of the sixcysteines conserved across the class of conventional defensins andresponsible for the three-dimensional structure necessary for theactivity of these peptides.

EXAMPLES Example 1 Identification of the Gene Encoding hDef-X

Isolation of BAC B0725 B12

To analyze the 8p23 region of the human genome, in particular in theregion known to carry genes encoding human defensins, a BAC (“BacterialArtificial Chromosome”) corresponding to said region was isolated. A BAClibrary covering the complete human genome was prepared from the ADN ofa human lymphoblastic line derived from individual No. 8445 of the CEPHfamilies. This line was used as source of high-molecular weight DNA. TheDNA was partially digested with the restriction enzyme BamHI, and thencloned at the BamHI site of the plasmid pBeloBacII. The clones thusobtained were “pooled” and screened according to the three-dimensionalanalytical procedure previously described for the screening of YAC(“Yeast Artificial Chromosome”) libraries (Chumakov et al., 1992 and1995). The three-dimensional pools obtained were screened by PCR withthe aid of the primers flanking the marker SHGC-10793, for Neutrophildefensin 4 precursor (GeneBank: accession number U18745); a clone of BACB0725 B12 was thus isolated.

After digestion with the restriction enzyme NotI, the size of the insertcarried by this BAC was determined on a 0.8% agarose gel after migrationby alternating field electrophoreses (CHEF) (4 hours at 9 volts/cm, withan angle of 100°, at 11° C. in 0.5×TAE buffer). It was thus demonstratedthat BAC B07025B12 carries an insert of 220 kb, with an internal sitefor the enzyme NotI.

Chromosomal Location of BAC B0725B12 by Fluorescent in situHybridization (FISH)

The chromosomal location of BAC in the candidate region 8p23.1-23.2 wasconfirmed by fluorescent in situ hybridization (FISH) on metaphasechromosomes, according to the method described by Cherif et al., (1990).

Sequencing of the BAC B0725B12 Insert

To sequence the BAC B0725B12 insert, a subclone library was preparedfrom the sonicated DNA of this BAC.

The cells derived from one liter of “overnight” culture were treated byalkaline lysis according to conventional techniques. Aftercentrifugation of the product obtained in a cesium chloride gradient, 12μg of BAC B0725B 12 DNA were purified. 3 μg of DNA were sonicated inorder to obtain fragments whose sizes are uniformly distributed from 1.2kb to 1.5 kb. The fragments obtained were treated in a volume of 50 μlwith 2 units of Vent polymerase for 20 minutes at 70° C., in thepresence of the 4 deoxytriphosphates (100 μM). The fragments withblocked ends resulting from this step were separated by electrophoreseson a 1% low-melting point agarose gel (60 volts for 3 hours). Thefragments grouped according to their sizes were excised and the bandsobtained treated with agarose. After extraction with chloroform anddialysis on Microcon 100 columns, the DNA in solution was adjusted to aconcentration of 100 ng/μl. A ligation was performed “overnight” bybringing 100 ng of fragmented DNA of BAC B0725B12 into contact with 20ng of DNA of the vector BluescriptSK linearized by enzymatic digestion,and treated with alkaline phosphatase. This reaction was carried out ina final volume of 10 μl in the presence of 40 units/μl of T4 DNA ligase(New England Biolabs). The ligation products then served to transform,by electroporation either a strain XL-Blue (for the multicopy plasmids),or a D10HB strain (for the subclones derived from BAC). The lacZ⁻ cloneswhich are resistant to the antibiotic were subcultured individually inmicroplates for storage and sequencing.

960 subclones corresponding to the insertion of fragments of 1.2 kb to1.5 kb at the BamHI site (made blunt) of the plasmid BluescriptSK.

The inserts of these subclones were amplified by PCR on bacterialcultures performed “overnight”, using the vector primers flanking theinserts. The sequence of the ends of these inserts (average 500 bases oneach side) was determined by automated fluorescent sequencing on an ABI377 sequencer equipped with the ABI Prism DNA Sequencing Analysissoftware (version 2.1.2).

The fragments having a sequence obtained from the sub-BACs wereassembled by the Gap4 software of R. Staden (Bonfield et al., 1995).This software allows the reconstruction of a complete sequence fromfragments of sequences. The sequence deduced from the alignment of thedifferent fragments is the consensus sequence.

Finally, directed sequencing techniques (systematic primer walking) wereused to perfect the sequences and to link the contigs.

Analysis of the Sequences for the Identification of Genes

The potential exons of the insert of BAC B0725B12 were located bysearching for homology on public protein, nucleic acid and EST(Expressed Sequence Tag) banks.

Databanks

Local refusions of the principal public banks were used. The proteinbank used consists of the nonredundant fusion of the libraries Genpept(automatic translation of GenBank, NCBI; Benson et al., 1996); Swissprot(George et al., 1996) and PIR/NBRF (Bairoch et al., 1996). The doubletswere eliminated by the “nrdb” software (public domain, NCBI; Benson etal., 1996). The internal repeats were then masked by the “xnu” software(public domain, NCBI; Benson et al., 1996). The resulting bank, calledNRPU (Non-Redundant Protein-Unique) served as reference for the searchesfor protein homologies. The homologies found with this bank made itpossible to locate regions potentially encoding a protein fragment whichis at least related to a known protein (coding exons). The EST bank usedis composed of the subsections “gbest” (1-9) of Genbank (NCBI; Benson etal., 1996). It contains all the fragments of public transcripts.

The homologies found with this bank made it possible to locatepotentially transcribed regions (present on the messenger RNA).

The nucleic acid bank (other than the ESTs) used contains all othersubsections of Genbank and of EMBL (Rodriguez-Tome et al., 1996) whosedoublets were eliminated as above.

Softwares

All the BLAST softwares (Altschul et al., 1990) for searching forhomologies between a sequence and protein or nucleic databanks wereused. The significance levels used depend on the length and on thecomplexity of the region tested as well as on the size of the referencebank. They were adjusted and adapted to each analysis.

Example 2 Analysis of the Nucleic and Peptide Sequences of hDef-X

Structure of the Gene Encoding hDef-X

The alignment of the gene encoding hDef-X with those encoding the knowndefensins made it possible to note a maximum homology between hDef-X andhDef-4 (FIG. 2). The overall level of homology between the two nucleicsequences is 72%. The only two regions of the genomic DNA of hDef-Xwhich do not exhibit homology with that of hDef-4 correspond to tworegions of insertion of a sequence which is repeated in the sequence ofhDef-X, which are absent from the sequence of hDef-4: one element of theAlu type (positions 2155 to 2335) and one fragment of element of Line 1(positions 2710 to 2780).

A high conservation of the region flanking in 5′ the promoter region isnoted, from which a high conservation of the elements for regulating thestability of the messenger and the expression of the gene probablyresults.

The high conservation of the sequence of exon 1, which is nottranslated, makes it possible to definitively attach the defensin hDef-Xto the class of hematopoietic conventional defensins, that is hDef-1, 2,3 and 4, in contrast to the enteric defensins hDef-5 and 6, whosegenomic sequence comprises only two exons, both of which are coding.

The alignment of the cDNAs for hDef-4 and hDef-X, indicating a homologygreater than 60%, is presented in FIG. 3.

Protein Analysis

The peptide sequence of the defensin according to the invention isrepresented in FIG. 4. The three domains of the protein are positionedas follows:

signal peptide: aa 1-19

pro region: aa 20-63

mature peptide: aa 64-94.

The specific degrees of homologies between hDef-4 and hDef-X werecalculated, according to the relevant region of the protein:

signal peptide: 63.2%

proregion: 52.3%

mature peptide: 37.9%.

The overall homology is 49.5%. These figures confirm the very highhomology which exists between defensins, a homology which is maximum atthe level of the signal peptides and minimum at the level of the maturepeptides.

The amino acids conserved in the class of conventional defensins arefound in the primary protein sequence of Def-X, in particular the sixcysteines involved in the three-dimensional structure thereof (FIG. 5).

In order to predict the secondary structures present on the defensinaccording to the invention, softwares for predicting secondary structurewhich are included in the Protein Interpretation Package, Copyright MRC1994, Medical Research Council, Hillsroad, Cambridge, United Kingdom,were used.

These softwares made it possible in particular to compare the predictedstructures of Def-X and HNP-4. Hydrophobicity profiles, alpha-helixstructures, β sheets, amphiphilicity are superposable in the twopeptides, which suggests similar processes for membrane insertion andfor formation of multimeric ion channels for these two defensins.

Example 3 Search for Mutations Associated with Familial Cancer Cases

Extraction of the Genomic DNA

The genomic DNA of immunodeficient or cancer patients is extracted fromthe peripheral venous blood after cellular lysis, protein digestion,organic partition and finally alcohol precipitation, according toconventional techniques well known to persons skilled in the art.

It is in particular advantageous to study the presence of mutations inthe genomic DNA of individuals coming from families with a high rate ofcancer, all types of cancer combined. A deficiency in a gene forgranulocyte defensin, such as hDef-X, can in fact have a role inpredisposition to cancers, as mentioned above.

Amplification of the Genomic DNA

Oligonucleotide primers are used for the genomic amplification of theexon sequences derived from BAC B0725B12; they are predicted by computeranalysis, and defined with the aid of the OSP software (Hillier et al.,1991).

All these primers contain, upstream of the bases specifically targetedby the amplification, a common universal oligonucleotide tail intendedto allow the sequencing of the amplified fragments (PU:5′-TGTAAAACGACGGCCAGT-3′ for the upstream primers, and RP:5′-CAGGAAACAGCTATGACC-3′ for the downstream primers).

The oligonucleotide primers are synthesized according to thephosphoramidite method, on a GENSET UFPS 24.1 synthesizer.

The amplification of each predicted exon sequence is carried out bypolymerase chain reaction (PCR), under the following conditions:

Final volume 50 μl Genomic DNA 100 ng MgCl2 2 mM dNTP (for each) 200 μMPrimer (for each) 7.5 pmoles AmpliTaq Gold DNA polymerase (Perkin) 1unit PCR buffer (10X = 0.1M Tris HCl pH 8.3, 0.5M KCl) 1 X.

The amplification is carried out in a Perkin Elmer 9600 or MJ ResearchPTC200 thermocycler with a heating cover. After heating at 94° C. for 10minutes, 35 cycles are performed. Each cycle comprises: 30 seconds at94° C., 1 minute at 55° C. and 30 seconds at 72° C. A final segment ofelongation of 7 minutes at 72° C. ends the amplification.

The quantity of amplification products obtained is determined on a96-well microplate, by fluorometry, using the Picogreen intercalatingagent (Molecular Probes).

Detection of the Polymorphisms/mutations

The products of genomic amplification by PCR are sequenced on the ABI377 automated sequencer using fluorescent primers labeled with ABIfluorochromes (Joe, Fam, Rox and Tamra) and Thermosequanase DNApolymerase (Amersham).

The reactions are carried out in 96-well microplates, on a Perkin Elmer9600 thermocycler, under conventional temperature cycle conditions:

8 cycles: denaturation: 5 sec. at 94° C.; annealing: 10 sec.; extension:30 sec. at 72° C., and then

13 cycles: denaturation: 5 sec. at 94° C.; extension: 30 sec. at 72° C.

6 units of Thermosequanase, and 5-25 ng of amplification product areused per sequence reaction.

At the end of the amplification cycles, the products of the sequencereactions are precipitated from ethanol, resuspended in loading buffercontaining formamide, denatured, and deposited on 4% acrylamide gels;the electrophoreses (2 hours 30 min at 3000 volts) are conducted on ABI377 sequencers equipped with ABI sofiwares for collection and analysis(ABI Prism DNA Sequencing Analysis Software, version 2.1.2.).

The sequences obtained in patients suffering from the deficienciesstudied, in particular in patients from families with a highpredisposition to cancers, are compared with the sequences obtained incontrol subjects, related or not related. A statistical analysis (lodscore calculation) makes it possible to conclude as to the significanceof the presence of a site of heterozygosity and to its association witha predisposition to cancers.

Example 4 Search for point Mutations

The point mutations identified as indicated above can then be detectedin patients having a potential deficiency in the gene encoding hDef-X,according to numerous methods known to persons skilled in the art. Amongthese, the following nonexhaustive lists may be mentioned:

sequencing

<<single nucleotide primer extension>> (Syvanen et al., 1990)

RFLP

search for <<single strand conformation polymorphism>>

methods based on a cleavage of the mismatched regions (enzymaticcleavage with S1 nuclease, chemical cleavage with various compounds suchas piperidine or osmium tetroxide)

detection of heteroduplex in electrophoresis

methods based on the use of <<allele specific oligonucleotide>> (ASO,Stoneking et al., 1991)

OLA method (<<dual color oligonucleotide ligation assay, Samiotaki etal., 1994)

ARMS (<<amplification refractory mutation system>>), or ASA (<<allelespecific amplification>>), or PASA (<<PCR amplification of specificallele>>) (Wu et al., 1989), method.

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6 4415 BASE PAIRS NUCLEOTIDE DOUBLE LINEAR DNA Homo sapiens Exon 11836..1874 Exon 2 3394..3577 Exon 3 4161..4380 start CDS 3406..3408 stopCDS 4276..4278 polyadenylation site 4374..4379 1 ACACCATTTG TCTTCATGTAACCCCATTAG CTATACCCTC TAGTGCAAGG AAACCATAGG 60 GCCTAGGTCA CACCATGAGGCTGCNCTTAC AAGTTATGCA AAAACTATGG ACTTGGGAGA 120 CCTGTGCGTA ACAACATCACACNCCAAATT TAACCAGCTC TCCCCATAAC AGCACGCTCA 180 TGTGTTACTG AGGAAATGCCTGTGGATTGG AGTGTGTTCT GTGTGCAGGA GGCTGGTCCA 240 GGTTTCACTT CTGCAGGACACTGGACGTTT CCCAAAACCA GCAGACTTTC CCCACGTGCA 300 CACACACCCC TTCTCATTTTGCCTCTACAT CCATATCCAC TGGGCCCTTC AGGCACCTAC 360 TAATGCCCTA GAACCTAAAACCATCATCTG GGGCCCAGTT CCCTGAATGG CCCTAATCTC 420 TTCCTCTGCT GGAATGAGTCCAGTGCCCAC TTCCTCCAAC GGTGAAATTG CTGGGCTGCT 480 ACAGATCAGG AACTCACTGCTTCCTCATAG GGGCAGCCGA CTTCACTGCT CTGCAACAGC 540 GACCACCCCT AGCGAGGCTTGAGATGCCTC TTGCCTCCTT AAGACTGAGG GAGACGCTTC 600 AGCTCTCACT CCACTGCCCCAAGTCCTCCA CAGCGCGGTG CCTGCTGCCT TCACACAGAG 660 CTGCAGGGGN AGGTCCTGTGTATCCGGCCT GCTGGACCAG CGCTGTGCAC AACCCTCCCA 720 TGGCAACAGT GGCTGCCCGGCCTGCACACT GGGCTTGGCA ACCTCGCTGT AGGTATTTAT 780 TCCCTCAGGA GTGACTGCATTCTTTTCCCA TTTCCAGAAA ACTGATGCCA TTTACCTCAC 840 TATGAGGAGG AGGAGGAGGAGGAGGGTGGA GAGTGGTACA TTTTAAAATG TGCACTATTC 900 TCCCTAGGAC TCCCCCTCAAATAACCCAGG AGGGACCATA CCAGCTCATT CCTGTGTATC 960 CCAAGCATAN GAGTAATCATCCCACTCATG CTGAGTGTAT GGTGGCCATT AAGCCTGCCC 1020 TGAACTGGCT TTAGAACAAGGTGTTTGAGC ACACAGCACC GTCTTGCTGC CACCTTGGCC 1080 CCCTCCCTTG TGAGACCTCTGAGACACATT NAGGTCTCAC CTAAAAATCT CAGGATTTCT 1140 AGGCCCAAAN CGGTCCTAAAAAATTGTTCA GTCTGAACTC TCTAAGGTCA AGAGAAGAGG 1200 TGGTTGCTCC CTCTAAGAAACCACATGTTG CATGTACATC CTTAATTCCG GAAAGTCCAA 1260 CAAACCTGCC CTGCTTAGCAACACAAGCCG AGGTGGTACT CCTCTCACCC GGGCATTCTC 1320 CAACACACCT GTTTGTCCAAACAGCTTTGA TTTGTTTTTA TAGTTGGACC CCAGGTTCCC 1380 AGGAGGCTGG TTCAGGCCATATTCCAAATC CTCATCTGTG TGTGAGTGGC ATTCTTAGCC 1440 TAGCCTCCTT ACAGGGTGGATACTATGATA CACAGCCAGG CTGTCCCAGT GGCTTTCAAT 1500 ATTCTTTTGG TCCAGATAGTTCAGCCTCAG CACCAGTGTA GGCATCACAG GGTCAATTGT 1560 CTTAGGAGTC ATGGAGAATTCATAGTTGGT AGCTACCTGG GCCTGGCCAG GGCTGACCAT 1620 AGACAAGGCA TCCCTCTGTGAACTCCTATT TTAATGCCAG CTTCCCAACA AATTTCTCAA 1680 CTGCTCTTAC CAGCAGGTATTTAAACTACT CAATAGAAAG TAACCCTGAA AATTAGGACA 1740 CCTGTTCCCA AAAGACCCTTAAATAGGGGA AGTCCTTTCN CTGCTTGTGC ACAGCTGCTG 1800 ATGTGGCAAC ATGAGGCCTGGGACAGGGGA CTGTCCTCTG CCCACTCTGG TAGCCTCACG 1860 TAGCTTAACA ATCTGTCAGTAATACAATAC AAAACTTAAA CTTTCATACT GCGGTTCCAC 1920 CCAGGAAGCT GTGTTCCCAATCTGACCCGT GATTATGGGG CCACCTCAGA GGGNACCCAG 1980 TGAGGGAATA TTTTGCCATCTGGGACTGTT GGTTGCTGGG GGCAGTGGCT ATGAGCTCAG 2040 TTAATAAACT CAAGCAGTTTCCTTCCAAAC ACACATGTCC TACTTAACGT GTCCAACAGA 2100 GATGATCATA CTCATANGCTGCTAAAACAT TANTTTTATT TTGAGAAAAG TCTATTCATG 2160 TTCTTGGCCC ATGGAGTTTTCATTTNATTA NTTTATTTAT TTTGCAGAGA TGGAGTCTCA 2220 CTATGTTGCT CAAGCTGGTCTCCAACTCCT GGGCTCAAGC GATCTTCCTA CTTTGGCCTT 2280 TGAAAGCGCT GAGATTGCCTGTGTGAGCCA TCATGGGGGC TCACTGGCCC ACTGATTAAT 2340 CAGATTAATT GTTTTTTGCTATTGAANTTG TTTGACTTCC TTGTATATTC GGATATTTAC 2400 CCATTCTAAC ACGTAGGGTTTGCAAATATT TTCTCTCATG TTCTGTGTTG CCTTTTCACT 2460 CAGTTGATGG TTTCCTTTGCTGTGCAGGTG CTTTAGTGTT CAACGCAGCC CCGCTTGTCT 2520 ATTTTCCATT TTATTGCCTGTCCCTTTGAT GTCATAGCCA AGAAATAATT GCCCAGATTA 2580 ATGTCAAAAA GCTTTATCCCTATATATTCT TCTAGTAGTT TATGGTTTCA GATCTTATGT 2640 TTAGGTCTTC AATCCATTGAGTTGATTTTT GTATGTGGTA TAAGAAAAAA GACCACATGT 2700 ATACATATCT CAAATTCTAAGGTAGTATAT ATTAGACACA TACAATGTGT CTATTTACAC 2760 ACATTGAGCT GAAAATAATAAACATATTTT TATCTTTCAA TCAACTCTAT CTCTATCTCA 2820 CTGAACTTGT TTCACCTATAGCCTGATGAG GTTGCTGTCC TCTCTACCCC AGCTCCTATA 2880 GGAGACTGCT CATCCCCTAACCTCAAAAAC CCCTTCATGA GGGTGATAAT GCCCTTGAAT 2940 CCTGCAATGA ATTAGTTCTCTACTACAGTG GAATTCAGGT CTGTTATGAG GGTCTGGATC 3000 TCTGAAGAGA AGAGCTCTCATTTTCAGAAA ATAAGCAGGA TTTATTCCCT GAAATTACTG 3060 AATTAAATCA CTGTTTCGATTACTTTTTGC AATATTAAAA GTAAATATTT AAACAGGTAA 3120 AAACAGAAAT AATGGTAGGGTCCTTATCAT CACCGTGAAT TCCAAGCTAG CATAGACACT 3180 AAACCTAGAG ATTCACACTAGAATGAAAGC TGGGAGAGCA GAGGAGTCTC AGAAGGATGT 3240 GGAGGCCAAT GGACACCTGCAACCTCTCCA ACGAAATGCC TACCTCCTCT CACTGCAGCA 3300 TCCATCTCTG AGCCTTCTCGCAGCAGAGCT ATAAATTCAG CCTGGCTCCT CCGTTCCCAC 3360 ACATCCACTC CTGCTCTCCCTCCTCTCCTC CAGGTGACTA CAGTTATGAG GACCCTCACC 3420 CTCCTCTCTG CCTTTCTCCTGGTGGCCCTT CAGGCCTGGG CAGAGCCGCT CCAGGCAAGA 3480 GCTCATGAGA TGCCAGCCCAGAAGCAGCCT CCAGCAGATG ACCAGGATGT GGTCATTTAC 3540 TTTTCAGGAG ATGACAGCTGCTCTCTTCAG GTTCCAGGTG AGAGATGCCA GCATGCAGAG 3600 CTACAGACTA GACAGAAGGACAGGAGACAG GCTCTGGAAT TGGATCTCAG TGGCAGATGT 3660 CACTTAGGTG GCTATACTTAACATCTCTGG TCCTGGATTT TCTCATATCT AAATGGAATA 3720 GAGAACCAAA GAAATCTAAGAGATTTTTCT TTCTCCAAAA ACTTGATTCC AAGATATGAC 3780 TGTGAAATTC ACTAGATTTAAGATATAAGG AGATGCTACC TAGTTCCTTC TGGAGCCAGA 3840 CAAACAAGCT TAAGTATATAGGAAAATATT TCACCCTGTC TATATAGGAG GTTTTAGAAC 3900 CTGGAGAGGA GCCTAAGAATGTGTTCAGGT GTGTGTGTGA TGGGCAGGAA TGCAGAAAAG 3960 TGAAGCAAAG GAGAATGAGTCTCGAATCCT GTGTGACCAG CACTGCTCTG TGTATTTATT 4020 CCTATTGACT GAGATTGTTTGTGCTACCGG CTGTAATACA GCCAACATCA CTCATCAGCC 4080 AACATGTGAC TTCTCCAAGATTCCCTTTAC CACCCACTGC TGNACCCCGT ACTCAGTTTC 4140 TGATGCTCTC TCTGGGTCCCCAGGCTCAAC AAAGGGCTTG ATCTGCCATT GCAGAGTACT 4200 ATACTGCATT TTTGGAGAACATCTTGGTGG GACCTGCTTC ATCCTTGGTG AACGCTACCC 4260 AATCTGCTGC TACTAAGCTTGCAGACTAGA GAAAAAGAGT TCATAATTTT CTTTGAGCAT 4320 TAAAGGGAAT TGTTATTCTTATACCTTGTC CTCGATTTCC TGTCCTCATC CCAAATAAAT 4380 ACTTGGTAAC ATGATTTCCGGGTTTTTTTT TTTTT 4415 453 BASE PAIRS NUCLEOTIDE DOUBLE LINEAR cDNA Homosapiens 2 CTCTGCCCAC TCTGGTAGCC TCACGTAGCT TAACAATCTG TGACTACAGT T ATGAGG 57 Met Arg 1 ACC CTC ACC CTC CTC TCT GCC TTT CTC CTG GTG GCC CTT CAGGCC TGG 105 Thr Leu Thr Leu Leu Ser Ala Phe Leu Leu Val Ala Leu Gln AlaTrp 5 10 15 GCA GAG CCG CTC CAG GCA AGA GCT CAT GAG ATG CCA GCC CAG AAGCAG 153 Ala Glu Pro Leu Gln Ala Arg Ala His Glu Met Pro Ala Gln Lys Gln20 25 30 CCT CCA GCA GAT GAC CAG GAT GTG GTC ATT TAC TTT TCA GGA GAT GAC201 Pro Pro Ala Asp Asp Gln Asp Val Val Ile Tyr Phe Ser Gly Asp Asp 3540 45 50 AGC TGC TCT CTT CAG GTT CCA GGC TCA ACA AAG GGC TTG ATC TGC CAT249 Ser Cys Ser Leu Gln Val Pro Gly Ser Thr Lys Gly Leu Ile Cys His 5560 65 TGC AGA GTA CTA TAC TGC ATT TTT GGA GAA CAT CTT GGT GGG ACC TGC297 Cys Arg Val Leu Tyr Cys Ile Phe Gly Glu His Leu Gly Gly Thr Cys 7075 80 TTC ATC CTT GGT GAA CGC TAC CCA ATC TGC TGC TAC TAA GCTTGCAGAC 346Phe Ile Leu Gly Glu Arg Tyr Pro Ile Cys Cys Tyr * 85 90 95 TAGAGAAAAAGAGTTCATAA TTTTCTTTGA GCATTAAAGG GAATTGTTAT TCTTATACCT 406 TGTCCTCGATTTCCTGTCCT CATCCCAAAT AAATACTTGG TAACATG 453 94 AMINO ACIDS AMINO ACIDSINGLE LINEAR PROTEIN Homo sapiens SIGNAL PEPTIDE 1..19 PRO REGION20..63 MATURE PEPTIDE 64..94 3 Met Arg Thr Leu Thr Leu Leu Ser Ala PheLeu Leu Val Ala Leu Gln 1 5 10 15 Ala Trp Ala Glu Pro Leu Gln Ala ArgAla His Glu Met Pro Ala Gln 20 25 30 Lys Gln Pro Pro Ala Asp Asp Gln AspVal Val Ile Tyr Phe Ser Gly 35 40 45 Asp Asp Ser Cys Ser Leu Gln Val ProGly Ser Thr Lys Gly Leu Ile 50 55 60 Cys His Cys Arg Val Leu Tyr Cys IlePhe Gly Glu His Leu Gly Gly 65 70 75 80 Thr Cys Phe Ile Leu Gly Glu ArgTyr Pro Ile Cys Cys Tyr 85 90 19 AMINO ACIDS AMINO ACID SINGLE LINEARSIGNAL PEPTIDE Homo sapiens 4 Met Arg Thr Leu Thr Leu Leu Ser Ala PheLeu Leu Val Ala Leu Gln 1 5 10 15 Ala Trp Ala 44 AMINO ACIDS AMINO ACIDSINGLE LINEAR PRO REGION Homo sapiens 5 Glu Pro Leu Gln Ala Arg Ala HisGlu Met Pro Ala Gln Lys Gln Pro 1 5 10 15 Pro Ala Asp Asp Gln Asp ValVal Ile Tyr Phe Ser Gly Asp Asp Ser 20 25 30 Cys Ser Leu Gln Val Pro GlySer Thr Lys Gly Leu 35 40 31 AMINO ACIDS AMINO ACID SINGLE LINEAR MATUREPEPTIDE Homo sapiens 6 Ile Cys His Cys Arg Val Leu Tyr Cys Ile Phe GlyGlu His Leu Gly 1 5 10 15 Gly Thr Cys Phe Ile Leu Gly Glu Arg Tyr ProIle Cys Cys Tyr 20 25 30

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising the sequence of SEQ ID No. 3;b) a polypeptide comprising an amino acid sequence at least 80%identical over the full length to the amino acid sequence of SEQ ID No.3; and c) a polypeptide comprising a fragment of at least 10 consecutiveamino acids of SEQ ID No. 3; wherein said isolated polypeptide has atleast one biological activity selected from the group consisting ofrecognition by an antiody specific for the polypeptide of SEQ ID NO: 3,antimicrobial activity, and cytotoxic activity.
 2. An isolatedpolypeptide comprising: a) a signal peptide comprising the sequence ofSEQ ID No. 4; b) a proregion comprising the sequence of SEQ ID No. 5; c)a mature peptide comprising the sequence SEQ ID No. 6; d) a polypeptidecomprising an amino acid sequence at least 90% identical over the fulllength to the amino acid sequence of SEQ ID No. 4, SEQ ID No. 5, or SEQID No. 6; or e) a fragment comprising at least 10 consecutive aminoacids of SEQ ID NO: 4, SEQ ID NO. 5, or SEQ ID NO. 6; wherein saidsignal peptide causes intra- or extracellular secretion of a polypeptideand/or is recognized by an antibody specific for the polypeptide of SEQID No. 3 or SEQ ID NO: 4; wherein said proregion inactivates theprecursor form of the defensin molecule and/or provides a support forthe acquisition of the active conformation of the mature peptide and/oris recognized by an antibody specific for the polypeptide of SEQ ID No.3 or SEQ ID NO: 5; wherein said mature peptide has at least onebiological activity selected from the group consisting of recognition byan antibody specific for the polypeptide of SEQ ID No. 3 or SEQ ID NO:6, antimicrobial activity, and cytotoxic activity; wherein said fragmentof SEQ ID No. 4 causes intra- or extracellular secretion of apolypeptide and/or is recognized by an antibody specific for thepolypeptide of SEQ ID No. 3 or SEQ ID NO: 4; wherein said fragment ofSEQ ID No. 5 inactivates the precursor form of the defensin moleculeand/or provides a support for the acquisition of the active conformationof the mature peptide and/or is recognized by an antibody specific forthe polypeptide of SEQ ID No. 3 or SEQ ID NO: 5; and wherein saidfragment of SEQ ID No. 6 has at least one biological activity selectedfrom the group consisting of recognition by an antibody specific for thepolypeptide of SEQ ID No. 3 or SEQ ID NO 6, antimicrobial activity, andcytotoxic activity.
 3. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising the sequence of SEQ ID No. 6;b) a polypeptide comprising an amino acid sequence at least 80%identical over the full length to the amino acid sequence of SEQ ID No.6; and c) a polypeptide comprising a fragment of at least 10 consecutiveamino acids of the sequence of SEQ ID No. 6 wherein said isolatedpolypeptide has at least one biological activity selected from the groupconsisting of recognition by an antibody specific for the polypeptide ofSEQ ID NO: 3 or SEQ ID NO: 6, antimicrobial activity, and cytotoxicactivity.
 4. The isolated polypeptide according to claim 1, wherein saidfragment comprises at least 15 consecutive amino acids.
 5. A compositioncomprising a pharmaceutically acceptable vehicle and an isolatedpolypeptide according to one of claims 1, 2, 3, or 4 in a form suitablefor topical administration.
 6. The isolated polypeptide according toclaim 1 comprising the sequence of SEQ ID No.
 3. 7. The isolatedpolypeptide according to claim 1, wherein said polypeptide comprises anamino acid sequence at least 80% identical over the full length to theamino acid sequence of SEQ ID No.
 3. 8. The isolated polypeptideaccording to claim 1, wherein said isolated polypeptide comprises afragment of at least 10 consecutive amino acids of the polypeptide ofSEQ ID No.
 3. 9. The isolated polypeptide according to claim 2,comprising a signal peptide comprising the sequence of SEQ ID No.
 4. 10.The isolated polypeptide according to claim 2, comprising a proregioncomprising the sequence of SEQ ID No.
 5. 11. The isolated polypeptideaccording to claim 2, comprising a mature peptide comprising thesequence of SEQ ID No.
 6. 12. The isolated polypeptide according toclaim 2, comprising an amino acid sequence at least 90% identical overthe full length to the amino acid sequence of SEQ ID No. 4, SEQ ID No.5, or SEQ ID No.
 6. 13. The isolated polypeptide according to claim 2comprising a fragment of at least 10 consecutive amino acids of thesignal peptide comprising the sequence of SEQ ID No.
 4. 14. The isolatedpolypeptide according to claim 2 comprising a fragment of at least 10consecutive amino acids of a proregion comprising the sequence of SEQ IDNo.
 5. 15. The isolated polypeptide according to claim 2 comprising afragment of at least 10 consecutive amino acids of a mature peptidecomprising the sequence of SEQ ID No.
 6. 16. The isolated polypeptideaccording to claim 3, wherein said isolated polypeptide comprises thesequence of SEQ ID No.
 6. 17. The isolated polypeptide according toclaim 3, wherein said isolated polypeptide comprises an amino acidsequence at least 80% identical over the full length to the amino acidsequence of SEQ ID No.
 6. 18. The isolated polypeptide according toclaim 3, wherein said isolated polypeptide comprises a fragment of atleast 10 consecutive amino acids of the sequence of SEQ ID No.
 6. 19.The composition according to claim 5, wherein said isolated polypeptidecomprises the sequence of SEQ ID No.
 3. 20. The composition according toclaim 5, wherein said isolated polypeptide comprises an amino acidsequence at least 80% identical over the full length to the amino acidsequence of SEQ ID No.
 3. 21. The composition according to claim 5,wherein said isolated polypeptide comprises a fragment of at least 10consecutive amino acids of the polypeptide of SEQ ID No.
 3. 22. Thecomposition according to claim 5, wherein said isolated polypeptidecomprises a signal peptide comprising the sequence of SEQ ID No.
 4. 23.The composition according to claim 5, wherein said isolated polypeptidecomprises a proregion comprising the sequence of SEQ ID No.
 5. 24. Thecomposition according to claim 5, wherein said isolated polypeptidecomprises a mature peptide comprising the sequence of SEQ ID
 6. 25. Thecomposition according to claim 5, wherein said isolated polypeptidecomprises an amino acid sequence at least 90% identical over the fulllength to the amino acid sequence of SEQ ID No. 4, SEQ ID No. 5, or SEQID No.
 6. 26. The composition according to claim 5, wherein saidisolated polypeptide comprises a fragment of at least 10 consecutiveamino acids of the signal peptide comprising the sequence of SEQ ID No.4.
 27. The composition according to claim 5, wherein said isolatedpolypeptide comprises a fragment of at least 10 consecutive amino acidsof a proregion comprising the sequence of SEQ ID No.
 5. 28. Thecomposition according to claim 5, wherein said isolated polypeptidecomprises a fragment of at least 10 consecutive amino acids of maturepeptide comprising the sequence of SEQ ID No.
 6. 29. The compositionaccording to claim 5, wherein said isolated polypeptide comprises thesequence of SEQ ID No.
 6. 30. The composition according to claim 5,wherein said isolated polypeptide comprises an amino acid sequence atleast 80% identical over the full length to the amino acid sequence ofSEQ ID No.
 6. 31. The composition according to claim 5, wherein saidisolated polypeptide comprises a fragment of at least 10 consecutiveamino acids of the sequence of SEQ ID No.
 6. 32. The isolatedpolypeptide according to claim 3, wherein said fragment comprises atleast 15 consecutive amino acids.
 33. The isolated polypeptide accordingto claim 1, wherein said antimicrobial activity is antifungal,antibacterial, antiviral, and/or antiparasitic.
 34. The isolatedpolypeptide according to claim 2, wherein said antimicrobial activity isantifungal, antibacterial, antiviral, and/or antiparasitic.
 35. Theisolated polypeptide according to claim 3, wherein said antimicrobialactivity is antifungal, antibacteral antiviral, and/or antiparasitic.36. The isolated polypeptide according to claim 2, wherein said fragmentcomprises at least 15 consecutive amino acids.
 37. The isolatedpolypeptide according to claim 1, wherein said biological activity isreognition by an antibody specific for the polypeptide of SEQ ID NO: 3.38. The isolated polypeptide according to claim 1, wherein saidbiological activity is antimicrobial activity.
 39. The isolaedpoypcptide according to claim 1, wherein said biological activity iscytotoxic activity.
 40. The isolated polypeptide according to claim 3,wherein said biological activity is recognition by an antibody specificfor the polypeptide of SEQ ID NO:
 3. 41. The isolated polypeptideaccording to claim 3, wherein said biological activity is antimicrobialactivity.
 42. The isolated polypeptide according to claim 3, whereinsaid biological activity is cytotoxic activity.
 43. The isolatedpolypeptide according to claim 2, wherein said signal peptide, orfragment comprising 10 consecutive amino acids thereof, causes intra- orextracellular secretion of a polypeptide.
 44. The isolated polypeptideaccording to claim 2, wherein said signal peptide, or fragmentcomprising at least consecutive 10 amino acids thereof, is recognized byan antibody specific for the polypeptide of SEQ ID No. 3 or SEQ ID No.4.
 45. The isolated polypeptide according to claim 2, wherein saidproregion, or fragment comprising at least 10 consecutive amino acidsthereof, inactivates the precursor form of the defensin molecule. 46.The isolated polypeptidc according to claim 2, wherein said proregion,or fragment comprising at least 10 consecutive amino acids thereof,provides a support for the acquisition of the active conformation of themature peptide.
 47. The isolated polypeptide according to claim 2,wherein said proregion, or fragment comprising at least 10 consecutiveamino acids thereof, is recognized by an antibody specific for thepolypeptide of SEQ ID No. 3 or SEQ ID No.
 5. 48. The isolatedpolypeptide according to claim 2, wherein said mature peptide, orfragment comprising at least 10 consecutive amino acids thereof, hasantimicrobial activity.
 49. The isolated polypeptide according to claim2, wherein said mature peptide, or fragment comprising at least 10consecutive amino acids thereof, has cytotoxic activity.
 50. Theisolated polypeptide according to claim 2, wherein said mature peptide,or fragment comprising at least 10 consecutive amino acids thereof, isrecognized by an antibody specific for the polypeptide of SEQ ID: No. 3or SEQ ID NO
 6. 51. The isolated polypeptide according to claim 12,wherein said isolated polypeptide has an amino acid sequence at least90% identical over the full length to the amino acid sequence of SEQ IDNo.
 4. 52. The isolated polypeptide according to claim 12, wherein saidisolated polypeptide has an amino acid sequence at least 90% identicalover the full length to the amino acid sequence of SEQ ID No.
 5. 53. Theisolated polypeptide according to claim 12, wherein said isolatedpolypeptide has an amino acid sequence at least 90% identical over thefull length to the amino acid sequence of SEQ ID No.
 6. 54. Thecomposition according to claim 25, wherein said isolated polypeptide hasan amino acid sequence at least 90% identical over the full length tothe amino acid sequence of SEQ ID No.
 4. 55. The composition accordingto claim 25, wherein said isolated polypeptide has an amino acidsequence at least 90% identical over the full length to the amino acidsequence of SEQ ID No.
 5. 56. The composition according to claim 25,wherein said isolated polypeptide has an amino acid sequence at least90% identical over the full length to the amino acid sequence of SEQ IDNo. 6.